Branching glycosyltransferase is an enzyme that catalyses the transfer of a segment of a 1,4. Burying the nonpolar surfaces in the interior of a protein creates a situation where the water molecules can hydrogen bond with each other without becoming excessively ordered. The fact that ofut1 possesses both fucosyltransferase and chaperone activity for notch raises the question of the respective contributions of these two activities to the genetic requirement for ofut1 in. Several glycosyltransferase genes including gly, gtf3, dgt1, galt2 are found located upstream of fap1, and genes coding for accessory secretion proteins such as secy2, gap1, gap2, gap3.
Rumi is a cap10 domain glycosyltransferase that modifies. Decouplingofrecombinantproteinproductionfrom escherichia. Review structures and mechanisms of glycosyltransferases. Cyan and yellow super fluorescent proteins with improved brightness, protein folding. In the er, glycosylation is used to monitor the status of protein folding, acting as a quality control mechanism to ensure that only properly folded proteins are trafficked to the golgi. Protein folding without any prior knowledge about protein structure is a difficult task. Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein. Despite advances in methods to produce defined glycoproteins by. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Wildtype gts may not have all the properties and functions that are desired for largescale production of carbohydrates. The glycosyltransferase hisdapgalnact2 is the key protein of the escherichia coli e.
Rapid proteinfolding assay using green fluorescent protein. The majority of therapeutic proteins are glycoproteins and therefore the posttranslational attachment of sugar residues is required. Each family gt family contains proteins that are related by sequence, and by corollary, fold. The glycosyltransferases have been classified into more than 90 families 7, 8. A new alpha helix motif required for protein glycosylation. The in vitro characterization of the catalytic activity of desvii, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in streptomyces venezuelae, is described. The modification is found in all domains of life, although it only exists in. Therefore, an important factor governing the folding of any protein is the distribution of its polar and nonpolar amino acids. This absence can be partially compensated by grp94 which, by recognizing advanced folding intermediate, collaborates with bip in assisting the folding pathway of selected substrates. Therefore, glycosylation may affect apob100s folding and maturation.
B glycosyltransferase reaction with a monophosphonucleotide leaving group can be coupled to a 5. Protein glycosylation is a common posttranslational modification that influences the functions and properties of proteins. A new alpha helix motif required for protein glycosylation 2 17. This first reported grampositive mgt shared significant homology with several mgts from gramnegative bacteria and the nterminal glycosyltransferase domain of class a highmolecularmass penicillinbinding proteins from different species. Glycopeptide specificity of the secretory protein folding sensor udp. Further investigations to study the in vivo activity of hisdapgalnact2 in e.
Characterization of glycosyltransferase desvii and its. Glycosyltransferase glt8d2 positively regulates apob100. Glycosyltransferase engineering for carbohydrate synthesis. Cyan and yellow super fluorescent proteins with improved. Peptide models of membrane protein folding arianna rath, david v. The gt and tp activities are coupled within the same class a pbps and also with their class b partners e.
A novel glycosyltransferase catalyses the transfer of glucose to glucosylated anthocyanins in purple sweet potato hongxia wang national key laboratory of plant molecular genetics, cas center for excellence in molecular plant sciences, institute of plant physiology and ecology, shanghai institutes for biological sciences, chinese academy of. The glycosyltransferase was isolated, the folding status of the hisdapgalnact2 was assessed and the activity of the protein in in vitro assays was shown. Landmark structure of cellulose synthase membrane protein complex. A glycosyltransferase reaction with a diphosphonucleotide leaving group can be coupled to an entpd, such as cd39l3. Caspofungin has been developed from the echinocandins and is in use as an antifungal agent. Yeast mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis alexander striebeck1, david a. These data indicate that by oglucosylating notch in the er, rumi regulates its folding andor trafficking and allows signaling at the cell membrane. This protein displays a similar type of fold than gta, but with some differences, so it can be considered as a new fold. Construction of a library of human glycosyltransferases. Contributions of chaperone and glycosyltransferase. Glycans and cellular functions i protein folding, er processing, quality control bcmb8. Also, the protein ofucosyltransferase ofut1 even acts as a chaperone for the folding and trafficking of its substrate glycoprotein notch.
Recombinant proteinbased therapeutics have become indispensable for the treatment of many diseases. Yeast mnn9 is both a priming glycosyltransferase and an. Polypeptide sequences can be obtained from nucleic acid sequences. Advances in understanding glycosyltransferases from a structural. Cell lysis escherichia coli leaky mutants recombinant protein. This protein was called ygt yersinia glycosyltransferase. Nlinked glycosylation plays a role in various cellular functions including protein folding, quality control, and secretion. Chaperone activity of protein ofucosyltransferase 1. Peptide models of membrane protein folding biochemistry.
Krishna,2 1 the johnson research foundation, department of biochemistry and biophysics, university of pennsylvania, philadelphia, usa 2 department of pharmaceutical sciences and biomolecular structure program, university of colorado health sciences center, denver, co, usa. M, the glycosyltransferase reaction is performed at 25 c for 24 h in 20. Protein folding is a remarkably complex physicochemical process via which a polymer of amino acids that samples many, many conformations in its unfolded state adopts a well. Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield 5. Glycosyltransferase definition of glycosyltransferase by. Recombinant protein based therapeutics have become indispensable for the treatment of many. In just three years, the green fluorescent protein gfp from the jellyfish aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. The large decrease in the abundance of ibranching on oglycan structures from stomach and colon tissues of mice lacking c2gnt2 suggests that in some tissues c2gnt2, and not ignt, is the dominant ibranching glycosyltransferase in protein o glycosylation. Paramecium bursaria chlorella virus1 encodes at least five putative glycosyltransferases that are probably involved in the synthesis of the glycan components of the viral major capsid protein. Gibbs free energy formula for protein folding 3 before the actual derivation is given, some basic facts should be stated, such as hydrophobicity, protein structures, and the environment in which the protein. The cgtase from bacillus stearothermophilus no2 possesses excellent catalytic properties but suffers from low production in e. A previous structural study established that the nterminal 211 amino acids of the a64r protein from pbcv1 form a gta group glycosyltransferase that is a retaining enzyme belonging to the gt34 family and that udpglucose possibly serves as the donor sugar. Deber division of molecular structure and function, research institute, hospital for sick children, toronto, ontario, canada m5g 1x8, and department of biochemistry, university of toronto, toronto, ontario, canada m5s 1a8. Glycobiology feb 23, 2009 complex carbohydrate research center.
Enterotoxigenic escherichia coli uses a nglycosyltransferase called etpc to modify the etpa protein, which is orthologous to hmw1a of haemophilus influenzae. Etpa operates as an adhesin that mediates the binding to intestinal epithelia and failure of glycosylation changes the. Our studies show that rumi is a protein oglucosyltransferase, capable of adding glucose to serine residues in notch egf repeats with the consensus c 1xsxpc 2 sequence. Irvine howard hughes medical institute, waksman institute and department of molecular biology and biochemistry, rutgers. To test the folding reporter, we expressed 20 different proteins from the hyperthermophilic archaeon pyrobaculum aerophilum 12, in e. Glycosyltransferases are found as soluble globular proteins 20, integral membrane bound proteins, or as lipidanchored enzymes 21 in. Some glycosyltransferase inhibitors are of use as drugs or antibiotics. Universal phosphatasecoupled glycosyltransferase assay. Protein misfolding is a common event in living cells. In this study, directed evolution was used to create three point mutants i631t. Nlinked glycans serve as quality control tags in the eukaryotic secretory pathway. Prosaii analysis showed that protein folding energy of our. In young and healthy cells, the misfolded protein load is disposed of by protein quality control pqc systems. Glycosyltransferases and transpeptidasespenicillinbinding.
Protein glycosylation in cancers and its potential therapeutic applications in neuroblastoma wanling ho1,2,3, wenming hsu4,5, minchuan huang5,6, kenji kadomatsu7 and akira nakagawara8 abstract glycosylation is the most complex posttranslational modification of proteins. In aging cells and in cells from certain individuals with genetic diseases, the load may overwhelm the pqc capacity, resulting in accumulation of misfolded proteins. Glycosyltransferase an overview sciencedirect topics. It generates z score of model, which is a measure of compatibility between its sequence and structure. Glycosyltransferase function in core 2type protein o. Protein glycosylation is a common co and posttranslational modification that has critical biological functions at both the cellular and wholeorganism levels. They are produced using wellestablished expression systems based on bacteria, yeast, insect and mammalian cells. Coexpression of folding accessory proteins for production of. Feb 17, 2016 the nterminal module is made of small domains and could be important for protein protein interactions in the pg synthesis complexes. The most common form of protein glycosylation is the attachment of carbohydrate moieties to asparagine residues hence nlinked glycosylation. This gene, which is one of the largest in the human genome, encodes a member of the nacetylglucosaminyltransferase gene family. Analysis of glycosylation motifs and glycosyltransferases. Ofut1 is a soluble er protein 10, 11 and, at least in drosophila, acts as a notch chaperone, facilitating the folding and secretion of notch. Help pages, faqs, uniprotkb manual, documents, news archive and biocuration projects.
Structural basis for the glycosyltransferase activity of the salmonella. Overall folds observed for glycosyltransferase enzymes. Protein structure prediction is often quoted as an np complete problem, i. Most glycosyltransferase enzymes form one of two folds. Cazy classifies enzymes in families based on sequence similarity, protein fold prediction and reaction mechanisms. Structural basis of the molecular ruler mechanism of a. Glycans and cellular functions i protein folding, er.
Sequencebased classification uses algorithmic methods to assign protein sequences to various families. It encodes a glycosyltransferase which participates in glycosylation of alphadystroglycan, and may carry out the synthesis of glycoprotein and glycosphingolipid sugar chains. The inorganic phosphate released by the coupling phosphatase may be detected using various. As a model membrane protein for these measurements we are using the transmembrane domain tmd of a putative glycosyltransferase fukutini fki, whose mislocalization has been linked to the onset of fukuyama muscular dytrophy. This driving force is called the hydrophobic effect, which can be described as the. Spatial organization and stoichiometry of nterminal domainmediated glycosyltransferase complexes in. There is another very important driving force for protein folding, however. In silico protein structure and function prediction. Glycosyltransferases gts, the enzymes that catalyse glycosidic bond. They catalyze the transfer of saccharide moieties from an activated nucleotide sugar also known as the glycosyl donor to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen carbon, nitrogen. Smallworld view of the amino acids that play a key role in protein folding.
Biological functions of glycosyltransferase genes involved. Apob100, the essential protein component of vldl 8, is a glycoprotein. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of sitespecifically controlling glycosylation at each of. Results rumi mutations cause a temperaturedependent loss of notch signaling we performed a chemical mutagenesis screen to identify novel. Glycosylation aids the folding of nascent polypeptide chains and stabilizes mature glycoproteins conformation 8. The model z score should be comparable to the z scores obtained from the template 20, 21. It was expressed in escherichia coli cells as a truncated protein. The formation of a membraneassociated k5 biosynthetic complex requires kfia, kfib, and. Find products for glycosyltransferase activity kits. Proteins and other charged biological polymers migrate in an electric field. We propose that lack of oglucosylation of notch in rumi mutants results in a ts defect in notch folding and signaling. Desvii is unique among glycosyltransferases in that it requires an additional protein component, desviii, for activity. Protein glycosylation in cancers and its potential. The extensive contacts between the helical domain and the gtb domains generate a unique funnelshapedgrooveadjacenttotheudphexosebindingsite,which was proposed to accommodate the acceptor protein 8.
In addition to the generally observed types of glycosylation, such as n glycosylation and mucintype o glycosylation, several unusual types of glycans have recently been discovered. Glycosyltransferase enzymes proteins and enzymes products. Ethambutol is an inhibitor of mycobacterial arabinotransferases and is used for the treatment of tuberculosis. Glycosylation was earlier thought to be restricted only to eukaryotes but now both o and n. Coexpression of folding accessory proteins, molecular chaperones, and human peptidylprolyl cistrans isomerase ppiase increased production of active cyclodextrin glycosyltransferase cgtase of bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant escherichia coli. Jan, 2015 recombinant protein based therapeutics have become indispensable for the treatment of many diseases. New roles for protein glycosylation in prokaryotes jerry eichler1, and michael koomey2 longheld to be a posttranslational modi. Pdf smallworld view of the amino acids that play a key. Only two structural folds, gta and gtb, have been identified for the nucleotide sugardependent enzymes, but other folds are now appearing for the soluble domains of lipid phosphosugardependent glycosyl transferases. Protein glycosylation has multiple functions in the cell. Rumi is a cap10 domain glycosyltransferase that modi. Clusters of lipid molecules only lipid portions at the edge of the cluster force the ordering of water.
Chaperone activity of protein ofucosyltransferase 1 promotes notch receptor folding tetsuya okajima, aiguo xu, liang lei, kenneth d. Glycosyltransferase structure and function springerlink. The same effect was also observed for other glycosyltransferases, such as fut9 table 5 and st3gal iv data not shown, suggesting that the pir protein mediated immobilization of human glycosyltransferases may be adversely affected by protein folding difficulties in the yeast endoplasmic reticulum. The glycosyltransferase pglh transfers three terminal nacetylgalactosamine galnac residues to a carrier, which is a prerequisite for bacterial protein n.
These snps, rs6976 and rs11177, lie in the 3utr of glycosyltransferase 8 domain containing 1 glt8d1 and in the coding region of guanine nucleotide binding protein like 3 gnl3 or nucleostemin, respectively. Structural studies, however, have only revealed two distinct glycosyltransferase gt folds, gta and gtb. Sweet new roles for protein glycosylation in prokaryotes. Rapid proteinfolding assay using green fluorescent. Here we report the results of a sitedirected mutagenesis approach to improve these fluorescent.
Glycosyltransferases catalyze glycosidic bond formation using sugar donors containing a nucleoside phosphate or a lipid phosphate leaving group. Expression of the functional recombinant human glycosyltransferase galnact2 in escherichia coli jennifer lauber1, rene handrick1, sebastian leptihn2, peter durre3 and sabine gaisser1 abstract background. Advances in understanding glycosyltransferases from a. Identification and characterization of a monofunctional. The role of the er glucosyltransferase in the quality. Pdf glycosyltransferase structure and function researchgate. A gene mgt encoding a monofunctional glycosyltransferase mgt from staphylococcus aureus has been identified. Glycosyltransferases were initially considered to be specific for a single glycosyl donor and acceptor, which led to the one enzymeone linkage concept. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Sequential glycosylation of proteins with substratespecific. Therefore, glycosylation may affect apob100s folding and maturation, which further affects vldl assembly and secretion.
Gta or gtb glycosyltransferases gtfs, gtfs are enzymes ec 2. Biological functions of glycosyltransferase genes involved in. The escherichia coli k5 capsule consists of a repeat structure of 4glca 1,4. Glycosyltransferases are found as soluble globular proteins 20, integral. Systems used to automatically annotate proteins with high accuracy. Fluorometric in situ monitoring of an escherichia coli. Nov 29, 2006 hodson n, griffiths g, cook n, pourhossein m, gottfridson e, lind t, lidholt k, roberts is 2000 identification that kfia, a protein essential for the biosynthesis of the escherichia coli k5 capsular polysaccharide, is an. Protein folding has been studied extensively in vitro, but much less is known about how folding proceeds in vivo. Cyclodextrin glycosyltransferases cgtases are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. Improved production of cyclodextrin glycosyltransferase. Sequence analysis revealed an nterminal enzyme domain, followed by a cysteine protease domain and a cterminal translocation and binding domain with tcdb. Fluorometric in situ monitoring of an escherichia coli cell. Characterization of the glycosyltransferase enzyme from.
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